The invention relates to the use of phosphinate-peptide analogs, as inhibitors of procollagen C-proteinase (PCP), for treating fibrotic diseases.
It is known that procollagen C-proteinase (PCP) is a key enzyme in fibrogenesis. It catalyzes the hydrolytic elimination of the procollagen propeptides from procollagens I, II, III and IV and also laminin V [cf. Amano S, Takahara K; Gerecke D, Nishiyama T, Lee S, Greenspan D S, Burgeson R E: Bone morphogenetic protein-1 is the processing enzyme for laminin 5 in human keratinocytes, Mol. Biol. Cell 7 (suppl.) 58A (1996)]. Consequently, PCP is a key enzyme in collagen processing [cf. Olsen B J: Morphogenesis: collagen it takes and bone it makes, Curr. Biol. 6: 645-647 (1996)]. It was demonstrated in BMP-I knock-out mice that complete absence of PCP leads to incomplete collagen processing with the deposition of atypical, loose collagen fibrils [cf. Suzuki N, Labosky P A, Furata Y, Hargett I, Dunn R, Fogo A B, Takahara K, Peters D M, Greenspan D S, Hogan B L: Failure of ventral body wall closure in mouse embryos lacking a procollagen C-proteinase encoded by BMP-1, a mammalian gene related to Drosophila tolloid, Development 122: 3587-3595 (1996)].
PCP is probably also responsible for the hydrolytic elimination of the propeptide sequence of lysyl oxidase. The elimination of the prosequence probably leads to the activation of the catalytic lysyl oxidase activity of the mature form [cf. Pachenko M V, Stetler-Stevenson W G, Trubetskoy O V, Gacheru S N, Kagan H M: Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase, J. Biol. Chem. 271: 7113-7119 (1996)]. Active lysyl oxidase covalently links opposing collagen fibrils to each other. In this way, PCP also indirectly increases the biological stability of the collagen towards degradation by collagenases.
PCP, or closely related proteins, appear also to play a role in the release of TGFxcex2-type growth factors. Recent studies have shown that PCP-like proteases are able to liberate TGFxcex2-type growth factors from an inactive complex with TGFxcex2-binding proteins [cf. Marques G, Musacchio M, Shimell M J, Wxc3xcmenberg-Stapleton K, Cho K W Y, O""Connor M B: Production of a DPP activity gradient in the early drosophila embryo through the opposing actions of the SOG and TLD proteins, Cell 91: 417-426 (1997); Blader P, Rastegar S, Fischer N, Strxc3xa4hle U: Cleavage of the BMP antagonist chordin by zebrafish tolloid, Science 278: 1937-1949 (1997)]. In this case, the binding partner for the TGFxcex2-type growth factors is decomposed by means of specific proteolysis. Consequently, PCP may possibly also indirectly possess a TGFxcex2-type agonistic activity. PCP can therefore be ascribed a crucial role in fibrogenesis.
PCP activity has its origin in splicing variants of the BMP-I gene [cf. Kessler E, Takahara K, Biniaminow L, Brusel M, Greenspan D S: Bone morphogenetic protein-1: The type I procollagen C-proteinase, Science 271: 360-362 (1996)); Reddi A H: BMP-1: Resurrection as procollagen C-proteinase, Science 217: 463 (1996); Li S W, Sieron A L, Fertala A, Hojima Y, Arnold W V, Prockop D J: The C-proteinase that processes procollagens to fibrillar collagens is identical to the protein previously identified as bone-morphogenetic protein-1. Proc. Natl. Acad. Sci. USA 93: 5127-5130 (1996)]. It is so far definitely known that splicing variants BMP-I-I and BMP-I-III (tld variant) are able to cut procollagen and pro-lysyl oxidase specifically. While relatively recent studies have identified further BMP-1 splicing variants, the biological function and substrate specificity of the latter have not yet been fully elucidated [cf. Janitz M, Heiser V, Bxc3x6ttcher U, Landt O, Lauster R: Three alternatively spliced variants of the gene coding for the human bone morphogenetic protein-1. J. Mol. Med. 76: 141-146 (1998)].
Although success has so far only been achieved in expression-cloning and purifying PCP in small yields, a large number of structural details of the enzyme are known. Thus, PCP belongs to the family of astacin proteases. The crystal structure of astacin is known in detail. There is a very high degree of structural homology between the catalytic domain of BMP-I and astacin such that it has been possible, on the basis of this homology, to assign to many amino acids in the PCP protease domain their probable structural and biochemical function [cf. Stxc3x6cker W, Gomis-Rxc3xcth F X, Bode W, Zwilling R: Implications of the three-dimensional structure of astacin for the structure and function of the astacin family of zinc-endopeptidases, Eur. J. Biochem. 214: 215-231 (1993)].
In the past, it has been possible, by means of computer-guided molecular modeling, to infer the binding of substate to the active center of the astacings in molecular detail [cf. Stxc3x6cker W, Grams F, Baumann U, Reinemer P, Gomis-Rxc3xcth FX, McKay DB, Bode W: The metzincinxe2x80x94Topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-edopeptidases, Prot Sci.: 823-840 (1995)]. These studies led to the rational design of phosphinate-peptide analogs which inhibit astacin with a high degree of potency. The complex between a phosphinate inhibitor and astacin has been strutually elucidated ([cf. Grams F, Dive V, Yiotakis A, Yiallouros I, Vassilou S, Zwilling R, Bode W, Stxc3x6cker W: Structure of astacin with transition-state analogue inhibitor, Nature Struct. Biol. 3: 671-675 (1996)].
Despite the high degree of structral homology between astacin and the catalytic domain of BMP-1, it has so far been assumed that, because of biochemical differences with regard to their reaction behavior and also in their ability to be inhibited by protease inhibitors, the two proteases are in fact markedly different. For example, there are biochemical differences between astacin and BMP-I with regard to their substrate specificity (as a crayfish digestive enzyme, astacin hydrolyzes collagen-like proteins relatively nonspecifically whereas PCP cuts highly specifically at only one site in the procollagen molecule and in pro-lysyl oxidase).
To date, only low-potency inhibitors of PCP, to which an antifibrotic effect is attributed, have been described in the literature [cf. Brenner M, Ho W B: C-proteinase inhibitors for the treatment of disorders related to the overproduction of collagen. WO 97/05865].
It has now been found, surprisingly, that phosphinate-peptide analogs of the general formula (I), 
in which
R1 represents hydrogen or methyl,
and their salts and isomers inhibit PCP with a very high degree of potency and can therefore be used for the treatment and prophylaxis of fibrotic diseases.
Within the context of the invention, preference is given to physiologically harmless salts. In general, physiologically harmless salts are salts of the compounds according to the invention with inorganic or organic acids. Preference is given to salts with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid, or salts with organic carboxylic or sulfonic acids, such as acetic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, or methanesulfonic acid, ethanesulfonic acid, phenylsulfonic acid, toluene-sulfonic acid or naphthalenedisulfonic acid.
The compounds according to the invention can exist in stereoisomeric forms which either relate to each other as image and mirror image (enantiomers) or which do not relate to each other as image and mirror image (diastereomers). The invention also relates to the antipodes and to the racemic forms as well as to the diastereomeric mixtures.
The compounds of the general formula (I) can be present in all the enantiomeric and diastereomeric forms. Preference is given to those isomers in which the parts of the molecule formed from proline, lysine and leucine possess the L configuration, as well as to their salts and prodrugs.
Particular preference is given to using phosphinate-peptide analogs of the general formula (I), which have the configuration as shown, 
in which
R1 represents hydrogen or methyl,
and their salts and isomers for the treatment and prophylaxis of fibrotic diseases.
Very particular preference is given to using the compound of the general formula (Ib) 
designated Z-PKF(PC)APL-O-Me in that which follows, and its enantiomers and its salts for the treatment and prophylaxis of fibrotic diseases.
The compounds of the general formula (I) are known and can in principle be prepared from corresponding phosphinate compounds using customary methods of peptide synthesis [cf., in this regard, Yiotakis A, Vassilio S, Jiracek J, Dive V: Protection of the Hydroxyphosphinyl Function of Phosphinic Dipeptides by Adamantyl. Application to the Solid-Phase Synthesis of Phosphinic Peptides, J. Org. Chem. 61: 6601-6605 (1996); Campagne J M, Coste J, Guillou L, Heitz A, Jouin P: Solid phase synthesis of phosphinic peptides, Tetrahedron Lett. 34: 4181-4184 (1993)].
The results were unexpected because BMP-I, meprin and astacin differ from each other, in particular, in the so-called S1xe2x80x2 loop. A sector from this loop is depicted here:
astacin: T-DPYD
BMP-I: KPPIG-Q
meprin: I-IG-Q
This loop forms the essential part of the S1xe2x80x2 binding pocket of the astacin-like proteases. It is here, as has been shown using the example of astacin and meprin, that the key to the differing substrate and inhibitor specificities of the two enzymes lies. Meprin and astacin differ very markedly from each other in the S1xe2x80x2 pocket. PCP additionally has two proline residues and one lysine residue in this region. As has been shown by computer modeling the protease domain of PCP, the lysine residue is very probably involved in binding the carboxyl group in the side chain of the aspartate in P1xe2x80x2 next to the cleavage site in the procollagen. According to the model, the proline residues are in the cis configuration and represent a peculiarity of the BMP-I subfamily among the astacins. By contrast, astacin does not cleave next to acidic residues. For these reasons, it was completely unexpected that a phosphinate which was designed for inhibiting astacin [cf Yiallouros I, Vassiliou S, Yiotakis A, Zwilling R, Stxc3x6cker W, Dive V: Phosphinic peptides, the first potent inhibitors of astacin, behave as extremely slow-binding inhibitors, Biochem. J. 331: 375-379 (1998)] would prove to be an effective PCP inhibitor.
The compounds of the general formula (I) according to the invention exhibit a valuable pharmacological activity spectrum which it was not possible to foresee.
Surprisingly, the compounds according to the invention are notable for having a very high potency when inhibiting PCP.
The compounds according to the invention are therefore suitable for treating liver fibroses of any genesis and fibroses which are manifested in other organs.
These fibroses include various groups of diseases which are accompanied by a qualitative change in collagen production or by an increased deposition of collagen in the extracellular space, such as liver fibroses of differing origin, such as alcoholic liver cirrhosis, biliary cirrhosis, hepatitis of viral or other genesis, idiopathic interstitial fibroses, idiopathic lung fibroses, acute pulmonary fibroses, acute respiratory distress syndrome (ARDS), perimuscular fibroses, pericentral fibroses, dermatofibromas, kidney fibroses, diabetic nephropathy, glomerulonephrites, systemic or local scleroderma, keloids, hypertrophic scar formation, joint adhesions, arthroses, myelofibroses, cicatrization of the cornea, cystic fibrosis, muscular fibroses, Duchenne""s muscular dystrophy, strictures of the esophagus, Omond""s disease, Crohn""s disease, ulcerative colitis and aneurysms of the large vessels.
In addition, the invention encompasses fibrotic diseases which are initiated or provoked by surgical scar revisions, plastic surgery, glaucoma, cataract fibroses, cicatrizations of the cornea, graft-versus-host disease, surgical interventions performed on tendons, nerve trapping syndromes, Dupuytren""s contracture, adhesions resulting from gynecological interventions, pelvic adhesions, epidural fibroses, and diseases of the thyroid gland or the parathyroid glands, and also by metastatic bone invasion, by multiple myeloma or by restenoses.
Detecting PCP Activity in a Fluorescence Sequencing Test
For the purpose of detecting PCP activity, a synthetic decapeptide substrate having the sequence
DABCYL-Asp-Phe-Tyr-Arg-Ala-Asp-Glen-Pro-Arg-Asp (EDANS)-NH2
was cleaved by adding PCP. This peptide frequency corresponds to the region in procollagen xcex12(I) which is cleaved by PCP. The cleavage sequence is known to biochemical experts [cf. Lee S T, Kessler E, Greenspan D S: Analysis of site-directed mutations in human pro-xcex12(I) collagen which block cleavage by the C-proteinase, J. Biol. Chem. 265: 21992-21996 (1990)], as is the dequenching test method [cf. Matayoshi E D, Wang G T, Krafft G A, Erickson J: Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer, Science 247: 954-958 (1989)].
The concentration of the synthetic peptide was 5.6 xcexcM, while the final buffer conditions were: 50 mM tris, pH 7.5, 150 mM NaCl and 0.005% Brij35.
The kinetic test was carried out in the following manner: the fluorescence substrate was dissolved in 90 xcexcl of reaction buffer. The kinetics of the conversion of the substrate by PCP were detected, in duplicate determinations, by measuring the fluorescence (ex. 355 nm/em. 538 nm) at from 0 to 120 min. The reaction was started by adding a suitable activity of PCP dissolved in 10 xcexcl of reaction buffer. The emission, as a measure of the proteolytic activity, was measured at time intervals of initially 5 min and then subsequently at longer time intervals. The spontaneous decomposition of the fluorescence substrate in the added presence of reaction buffer lacking PCP activity was measured as the negative control. The fluorescence measurements took place at 37xc2x0 C. over a period of approx. 4 h. After the reaction had come to an end, the substrate was completely decomposed by adding proteinase K (Boehringer Mannheim, 1.44 xcexcg per reaction mixture, dissolved in 10 xcexcl of PBS) and incubating at 37xc2x0 C. for 20 min. The fact that the hydrolysis was complete was shown by there being no further increase in the fluorescence with time.
The relative conversion of substrate in % of the total quantity was calculated from:
% conversion=(Ftxe2x88x92Fxe2x88x92t)÷(Ftotalxe2x88x92Fin1)xc3x97100%
where Ft is the relative fluorescence after a time interval t while incubating with PCP. Fxe2x88x92t is the corresponding relative fluorescence after the time interval t without PCP being added, Ftotal is the fluorescence following total hydrolysis produced by adding proteinase K, and Fin1 is the initial relative fluorecence before starting the reaction by adding proteinase K.
In the test, the PCP activity used was typically adjusted such that approx. 20% of the substrate was converted by inhibited enzyme within the measurement period.
FIG. 1 shows typical reaction kinetics (% conversion) which were obtained when adding the phosphinate inhibitor Z-PKF(PC)APL-O-Me.
The percentage inhibition obtained in the added presence of the inhibitors was calculated as follows:
% inhibition=100%xc3x97% conversion (with inhibitor)+% conversion (without inhibitor)
FIG. 2 depicts the concentration-effect relationship of a phosphinate inhibitor.
Determining the Specificity of PCP Inhibitors by Carrying Out in-vitro Investigations Using the Astacin-like Protease Meprin
Meprin is an enzyme which belongs to the astacin protease family and which is found in humans (Stxc3x6cker W, Gomis-Rxc3xcth F X, Bode W, Zwilling R: Implications of the three-dimensional structure of astacin for the structure and function of the astacin family of zinc-endopeptidases, Eur. J. Biochem. 214: 215-231 (1993)). The meprin activity test was carried out in a completely analogous manner to the in-vitro assay for measuring PCP activity. Instead of the recombinant PCP, use was made of human meprin, which also transforms the fluorescence-labeled decapeptide.
FIG. 3 shows the specificity of the highly active phosphinate-peptide analog Z-PKF(PC)APL-O-Me for PCP. Even at concentrations of up to 100 nM, meprin is only slightly inhibited (in a dose-dependent manner) in its transformation of the fluorescence-labeled peptide.
Demonstration of Biological Activity
The biological activity of the substances can be demonstrated in cell culture assays and in vivo. For example, after administering the inhibitors, it is possible, in human cell lines, to measure the decline in the concentration of free procollagen xcex11(III) propeptide in the supernatants, because this peptide is released by PCP activity. The PIIICP concentrations in the supernatant can be measured using a recently established assay [cf. Burchardt E R, Schrxc3x6der W, Heke M, Kohlmeyer J, Neumann R, Kroll W: Expression cloning of C-terminal procollagen (III) propeptide and its use in a novel serum assay to monitor liver fibrogenesis, Hepatology 26: 487A (1997)].
In order to demonstrate the antifibrotic effect of the substances in the liver, it is possible, for example, to use the animal model of acute [cf. Johnson S J, Hines J E, Burt A D: Phenotypic modulation of perisinusoidal cells following acute liver injury: a quantitative analysis. Int. J. Exp. Path 1992; 73: 765-772 (1992)] or chronic [cf. McLean E, McLean A, Sutton P: Instant Cirrhosis. An improved method for producing cirrhosis of the liver in rats by simultaneous administration of carbon tetrachloride and phenobarbitone, Br. J. Exp. Pathol. 1969; 50: 502-506 (1969)] carbon tetrachloride-induced liver damage, the model of liver fibrosis due to bile duct ligature [cf. Kountouras J, Billing B, Scheuer P: Prolonged bile obstruction: a new experimental model for cirrhosis in the rat, Br. J. Exp. Pathol. 1984; 65: 305-311 (1984)] or the liver fibrosis which is induced by hetemlogous serum [cf. Bhunchet E, Wake K.: Suppression of experimental hepatic fibrosis by administration of vitamin A, Lab. Invest 1985; 52: 182-194 (1984)]. It is also possible to use other animal models in which liver fibrosis occurs for demonstrating the antifibrotic effect.
Depending on the organ in which the fibrosis is manifested, or on the nature of the fibrotic damage, it is also possible to use animal models for other manifestations of fibrosis, for example in the heart, in the kidneys, in the lungs, in the skin or in other organs.
The reduction in collagen deposition can be demonstrated, for example, by determining the content of hydroxyproline [cf. Gerling B, Becker M, Waldschmidt J, Rehmann M, Schuppan D.: Elevated serum aminoterminal procollagen type-III-peptide parallels collagen accumulation in rats with secondary biliary fibrosis, Hepatology 1996; 25: 79-84 (1996)] in the fibrotic organs or by means of quantitative morphometry [cf. Kauschke S G, Knorr A, Olzen M, Burchardt E R: Expression of collagen (III) as determined by quantitative PCR and its correlation with extracellular collagen deposition in the rat CCl4 model of liver fibrosis. Hepatology 26: 538A (1997)].